The present invention relates to a process for treating collagenous connective tissue or structural support tissue removed from an animal donor for implant into a recipient without an immune and inflammatory rejection. Collagenous connective tissue or structural support tissue may be heart valve tissue, blood vessel, pericardium, omentum, fascia, tendon, ligament, intestine, cartilage, bone, membrane, or other such tissue.
Implants from animal donors into a human recipient to correct defective body components are known in the prior art. For instance, hemostatic collagen implants and collagen injections have been used for hemostasis and tissue augmentation; homograft and xenograft tissue heart valves have been widely implanted with variable results. The implant materials for these procedures were derived from animal origins that contained collagen, elastin, pericardium, and cells along with the proteins and other substances within the extracellular matrices. In the prior art the treatment of the implant depended upon its intended use. Implant materials generally were either intended for temporary implant or for permanent incorporation by the recipient. As an example of a temporary implant, hemostatic collagen may be used for emergencies to arrest blood loss; once hemostasis occurs, there is no further utility for the collagen. The concern where the implant is temporary is that major complications such as infection, pyrogenic shock or a major foreign body reaction by the recipient might occur. Another example of a temporary implant is the absorbent suture which is intended for temporary use so as to permit a wound to heal as the tissue regains its strength. Where the temporary implant material is derived from an animal, the implant material need not be homogeneous; it can be denatured and slightly toxic to the recipient.
Implant materials intended for permanent or long lasting implantation may provide structural support for a body part or may be an active functional organ such as a kidney, liver, or heart. For active functional organs, an immune compatible organ is most desirable. Preferably, it is harvested from a donor whose tissue closely matches that of the recipient.
In the case of permanent structural support, implants derived from biological sources are treated in the prior art before implantation utilizing one of three basic strategies.
The first basic strategy involves chemical modification, i.e., chemical modification of functional groups of tissue components, crosslinking, surface modification, etc. It can also increase the mechanical strength as well as the durability of the tissue by adding crosslinking to tissue fibers. Chemical modification can reduce the antigenicity and alter other properties of the tissue when it is transplanted into a different species. One of the most successful modifications of connective tissue for use as a bioprothesis is the glutaraldehyde fixation or crosslinking of porcine heart valves and bovine pericardium. However, there are limitations on this technology. Glutaraldehyde treated tissue has been shown to be toxic and host cells will not infiltrate tissue subjected to this treatment. The physical properties of glutaraldehyde treated tissue are very different from the physical properties of native untreated tissues. In the case of glutaraldehyde treated tissue for a heart valve prosthesis, calcification is known to be a major cause of valve failure. Numerous methods have been developed to modify the physical properties of glutaraldehyde treated tissue to overcome these limitations. These methods include but are not limited to the following:
a) treatment with detergent or surfactant after glutaraldehyde crosslinking;
b) covalently binding diphosphonates to the glutaraldehyde treated tissue;
c) covalently binding amino-substituted aliphatic functional acid to the glutaraldehyde treated tissue;
d) covalently binding sulfated polysaccharides, especially chondroitin sulfate after glutaraldehyde crosslinking;
e) treatment with ferric or stannic salts either before of after glutaraldehyde crosslinking;
f) incorporation of polymers, especially elastomeric polymers, into the glutaraldehyde treated tissue; or
g) immersing glutaraldehyde treated tissues in solutions of a water-soluble phosphate ester or a quaternary ammonium salt or a sulfated higher aliphatic alcohol.
None of these methods, however, has been entirely successful.
The second basic strategy for long-term implantation involves making a piece of tissue with no vital cells. Some examples of this are the following:
a) Decellularization (killing cells by hypotonic shock then followed with nucleases). Goldstein, U.S. Pat. Nos. 5,613,982; 5,632,778; 5,899,936 and 5,843,182;
b) Controlled autolysis Jaffe, U.S. Pat. Nos. 5,843,180; 5,843,181 and 5,720,777;
c) Killing of cells by radiation, as shown in Schinstein, U.S. Pat. Nos. 5,795,790; 8,843,431; 5,843, 717 and 5,935,849; and Badylak, U.S. Pat. No. 6,126,686); peracetic acid (Badylak U.S. Pat. No. 6,126,686);
d) Acid treatment (Abraham, U.S. Pat. No. 5,993,833); and
e) Other processes in the prior art construct composites of purified collagenous material with a synthetic scaffold (Bell, U.S. Pat. No. 6,051,750). The objective of all of these methods is to create a matrix that the host can accept such that cells in the recipient's body can migrate into the matrix and eventually remodel the material into a living tissue.
The third strategy involves the preservation or incorporation of living cells in the transplanted material. Tissues removed from the donors have a finite time limit before cells in the tissues die due to autolysis as a result of lack of oxygen. Conditions such as lowering the temperature and placing the tissues in solutions containing ischemic protection agents can keep cells vital for an extended time. Cryopreservation has been used to preserve the vitality of cells in tissues to an even longer time. However, the number of cells that survive in a host is substantially affected by the time lapse between harvesting and cryopreservation, and by the length of time the tissue has been preserved, by the thawing process and the implant procedures. The surviving cells will thereafter face a very hostile environment in the host unless there is a close genetic match or there is initiated an immune suppressive therapy.
The treatment of scaffold material to form living tissue outside the host has become a popular research topic. In an incubation environment, in order to have enough cells to infiltrate and grow in the scaffold material, the cells must be able to or stimulated to divide and grow rapidly.
To transition from a growing to a stable phase, such rapidly dividing and growing cells must be controlled. Once the cells are controlled, whether they will remain controlled after implant in the host is a major concern under this third strategy. The basic limitation that pervades this third strategy of the prior art is the lack of a sterilization method that can preserve living mammalian cells.
While the third strategy either keeps the cells from the donor alive or utilizes facilities outside of the recipient's body to culture or incubate living cells into the implants, by contrast the second strategy utilizes the recipient's cells as the donor source for cells and the recipient's body environment as an incubator. The current invention provides a process where implant materials will interact with the recipient's body without an immune and inflammatory rejection and the body cells will migrate to the implant to remodel the implant into living tissue.
Immune rejection is a major problem in transplantology. The major cause of immune rejection is the difference between the cell surface molecules of the donor and the recipient. If the implant contains donor living cells, this problem is prolonged and amplified as long as the donor cells are living and proliferating. If the implanted tissue does not contain living cells, the dead cells still present a problem. For this reason, methods have been developed to extract the remnants of dead cells from tissue through the use of detergents, solvents, etc. Such methods require extraction to be very aggressive to ensure that all the unwanted materials are extracted. Under such harsh conditions, tissue matrix components which are critical to the integrity of the tissue are also destroyed. The destruction of the tissue matrix components can be subtle and difficult to detect and often not easily observed using light microscopy or electron microscopy.
It has been suggested in the prior art that polyethylene glycol (PEG) may reduce immune response of recipients to allogeneic transplants (“Heart Preservation Solution Containing Polyethylene Glycol: An Immunosuppressive Effect” by Collins, et al. in Lancet, 338:390 (1991)). In one study a 30% reduction has been observed in the incidence of acute rejection in a group of heart transplant recipients in which the donor organ had been stored at 4° C. in a solution containing 5% PEG. In a subsequent study, PEG produced a modest but statistically significant increase in rat liver allograft survival time from 9.6 to 11.9 days (see “The Immunosuppressive Effect of Polyethyene Glycol in a Flush Solution for Rat Liver Transplantation” by Tokunaga, et al. in Transplantation, 54:756-8 (1992)). In these studies, the transplant organ was merely soaked in the PEG solution without subsequent cryopreservation. In U.S. Pat. No. 4,938,961, Collins, et al., discloses an organ preservation solution containing polyethylene glycol, along with a variety of further ingredients including: 30-40 mM NaOH, 100 mM lactobionic acid, 25 mM KH2PO4, 10 mM KOH, 30 mM raffinose, and 3 mM glutathione. This solution is used for the transport of an organ from a donor to a recipient. U.S. Pat. No. 6,280,925 to Bruckbank discloses a tissue pretreatment solution containing PEG and glutathione for use prior to cryopreservation. In the current invention, by incubating PEG with tissues under a high ionic strength condition, the collagen molecules remain insoluble but the interaction between proteoglycans and collagens is weakened. In this manner, proteoglycans on the surface of collagen fibers are replaced by PEG while preventing the collagen fibers from collapsing and aggregating.
While it is important to remove and mask substances antigenic to the recipients, it is also important to consider other factors relevant to the behavior and survival of the implanted materials. Acute and chronic inflammation are major defense mechanisms our bodies use against foreign invading materials as well as removing damaged materials resulting from injury or cell death. The mechanisms are initiated by inflammatory cells arriving at the site of foreign material. They digest the foreign material by oxidation. The digested material or the digestion process further recruits more inflammatory cells. As a result, the cycle continues until all the foreign or damaged “wounded self” materials are removed. Very often, an acute inflammatory reaction can cause damaged tissues to be even more vulnerable to further recruitment of inflammatory reaction thereby resulting in chronic inflammation. An example is chronic arthritis; inflammatory cells continue to attack an already inflamed joint matrice from previous injury or disease. The inflamed tissues recruit a further inflammatory reaction which damages the joint matrice even further and results in a vicious cycle that leads to a permanent disabling of the joint. In most cases, inflammatory reactions subside as the injured tissue or foreign bodies are cleared from the recipient. In the field of implantable biomedical implants, long term success of an implantable biomaterial depends on the host prevention of the inflammatory reaction against the implant.
In the present invention, the foreign implant becomes acceptable to the host or recipient by a process that renders the implant less oxidizable and thus less inflammatory. This is achieved in part by using hydrogen peroxide to oxidize the foreign implant.
Thus, the present invention provides a process for treating collagenous connective tissue such that the tissue is implantable without an immune and inflammatory rejection. The process permits the collagen matrix to remain intact and permits the residual antigenic components to be masked by polyethylene glycol; the process permits collagen fibers to remain structurally viable and the tissue to be oxidized to reduce recruitment of inflammatory cells; the process permits the tissue to incorporate water insoluble anti-inflammatory agents that inhibit the arrival of inflammatory cells at the implant site, and permits the tissue fibers to absorb an anti-thrombosis agent on their surface.